Cocaine activates reward pathways in the brain involving dopaminergic neurons in the striatum. Nagai et al. and Cahill et al. both discovered that Rap1, a guanosine triphosphatase (GTPase), mediated cocaine-induced signaling events that alter synaptic activity of medium spiny neurons in the nucleus accumbens. Using mouse striatal slice preparations exposed to either a dopamine type 1 receptor (D1R) agonist (SKF81297) or an activator of adenylyl cyclase (forskolin) to increase cyclic adenosine monophosphate (cAMP) concentrations, Nagai et al. applied a phosphoproteomic approach in which proteins phosphorylated on serine or threonine residues were enriched by affinity purification with 14-3-3–coupled beads and then analyzed by mass spectrometry. This analysis identified 47 proteins that exhibited an increase in phosphorylation in response to both stimuli, and included the Rap1 guanine nucleotide exchange factor (GEF) Rasgrp2. In vitro kinase assays with protein kinase A (PKA) confirmed that Rasgrp2 was a direct PKA substrate. Exposure of striatal slices to either SKF81297 or forskolin increased the amount of GTP-bound (active) Rap1, consistent with phosphorylation of Rasgrp2 stimulating its GEF activity. Giving mice a single dose of cocaine also increased Rasgrp2 phosphorylation and GTP-bound Rap1 in the nucleus accumbens. Viral-mediated expression of a constitutively active form of the catalytic subunit of PKA or an active form of Rap1 specifically in D1R-expressing neurons increased neuronal excitability and increased reward behavior (conditioned place preference after 3 consecutive days of cocaine injections). Localized knockout of Rap1 in the nucleus accumbens reduced neuronal excitability and cocaine-induced reward behavior.

The microRNAs miR-132 and miR-212 are induced by cocaine, and Cahill et al. identified PDZ-RhoGEF, which is a GEF for RhoA, as a top predicted target of this pair of miRNAs. Knocking down these miRNAs both enhanced cocaine-induced reward behavior and increased the abundance of PDZ-RhoGEF. In nucleus accumbens isolated from mice 24 hours after cocaine injection or self-administration for 7 days, the amount of PDZ-RhoGEF in the crude cytosolic fraction decreased and the amount in the crude nuclear fraction increased. In contrast, 4 weeks after the last dose of cocaine, PDZ-RhoGEF was more abundant in the cytosolic fraction than the nuclear fraction of the nucleus accumbens. Twenty-four hours after the 7-day cocaine injection paradigm (24-7), GTP-bound RhoA in the nuclear fraction was increased, which was associated with an increase in the binding of the serum response factor (SRF) coactivator MAL to filamentous actin in the nucleus, an indicator of increased SRF-mediated transcription. Rap1b was among the SRF-MAL target genes that exhibited increased expression in the nucleus accumbens in response to cocaine in the 24-7 paradigm. Cocaine induced an increase in Rap1b, which was diminished in mice with a nucleus accumbens–specific knockdown of SRF and was absent 2 weeks after the mice received the last cocaine dose. Rap1 activates phosphoinositide 3-kinase (PI3K) and Rap1 coimmunoprecipitated with PI3K in the nucleus accumbens, even in mice not exposed to cocaine. Consistent with Rap1b activating PI3K, which then stimulates the kinase Akt and the translation-promoting complex mTORC1, phosphorylation of Akt and mTOR increased in the nucleus accumbens of mice administered cocaine (24-7 paradigm) and also in mice overexpressing Rap1b in the nucleus accumbens. Rap1 overexpression in the nucleus accumbens produced an increase in the number of thin spines (considered immature), an effect blocked by coexpression of dominant-negative Akt. Furthermore, expression of dominant-negative Akt or viral-mediated knockout of Rap1 in the nucleus accumbens inhibited cocaine-induced increase in thin spines. This Rap1-PI3K-Akt-mTORC1 pathway also contributed to cocaine-induced reward behavior and locomotor activity.

This pair of studies examines the early signaling events associated with short-term cocaine administration and the later events associated with long-term cocaine administration. Nagai et al. connected D1R signaling through cAMP and PKA to increased activation of Rap1 through Rasgrp2 in response to acute cocaine administration. Cahill et al. connected long-term cocaine-induced changes in gene expression to an increase in Rap1b that altered spine morphology through the PI3K-Akt-mTORC1 pathway. The Rap1-dependent changes reported by Cahill et al. eventually reversed several weeks after the last cocaine dose.